An important aspect of the production of recombinant (genetically engineered) peptides, including oligo- and polypeptides, notably proteins, intended for therapeutic use in humans or animals is purification of the peptides in question to a sufficiently high level of purity, in particular such that the desired protein is essentially completely free of contamination with, in particular, (a) any extraneous proteins which may arise in the production process (typically a fermentation process or the like employing a selected or genetically modified strain of an appropriate microorganism) and (b) undesirable metal ions (notably heavy-metal ions) that may have been introduced in the course of the production process.
Immobilized metal ion affinity chromatography (IMAC) is a versatile separation procedure that exploits differences in the affinities exhibited by many biopolymers for metal ions. The technique involves the chelation of a suitable metal ion onto a solid support matrix whose surface has previously been chemically modified with a polydentate ligand. The resulting immobilized metal ion chelating complex then has the potential to coordinate with one or more electron donor groups resident on the surface of the interacting protein (Sulkowski, E., Trends in Biotechnology, 3 (1985) 1-6; Porath, J., Carlsson, I., Olsson, I. and Belfrage, G., Nature, 258 (1975) 598-599; Kagedal, L., in “Protein Purification” (Ed., J. C. Janson, and L. Ryden), VCH Publishers (1989) pp. 227-251; Zachariou, M. and Hearn, M. T. W., Biochemistry, 35 (1996) 202-211. Separation selectivity is then achieved on the basis of differences in the thermodynamic stabilities of the adsorbed protein/immobilized metal ion complexes. Proteins whose adsorption complexes are the least stable will be eluted first, whilst proteins that form more stable complexes will be eluted later. The greater the difference in the equilibrium association constants, i.e. the larger the differences in the dissociation constants (KD) of the respective protein/immobilized metal ion coordination complexes, the higher the resolution obtained. Consequently, the amino acid composition, surface distribution of particular amino acid residues, as well as the conformation of the protein all play important roles in determining the affinity of a protein for a particular IMAC system. As a result, proteins with very similar properties with respect to charge, molecular size and amino acid composition, but with differences in their tertiary structures, may be resolved.
Most of the research interest into the use of IMAC over the past 20 years has revolved around the application of 1st row transition metal ions of borderline hardness (vide infra), such as Cu2+, Zn2+ and Ni2+. These metal ions demonstrate intermediate metal ion stability constants, e.g. logo values between 5 and 10, for both aromatic and aliphatic amines, as well as for carboxylate functional groups (Wong, J. W., Albright, R. L. and Wang, N. H. L., Separation and Purification Methods, 20 (1991) 49-57; Zachariou, M., Traverso, I., Spiccia, L. and Hearn, M. T. W., Journal of Physical Chemistry, 100 (1996) 12680-12690). A number of unconstrained tridentate chelates that exhibit these binding properties with M2+ ions can be chemically immobilized onto support materials. Despite their limitations with regard to the magnitude of the corresponding log β values and their resulting relatively low selectivity capabilities, unconstrained types of chelating compounds such as iminodiacetic acid (IDA) constitute the principle types of chelating ligand employed hitherto in such IMAC investigations [see, e.g., Kagedal, L., in “Protein Purification” (Eds. J. C. Janson and L. Ryden), VCH Publishers (1989) pp 227-251]. Applications illustrative of the use of immobilized M2+-IDA-based IMAC systems include the purification of α-amylases from germinated wheat using immobilized Cu2+-IDA [Zawistowska, U., Sangster, K., Zawistowski, J., Langstaff, J. and Friessen, A. D., Cereal Chemistry, 65 (1988) 5413-5418]; and purification of human clotting factor VII [Weeransinghe, K. M., Scully, M. F. and Kadder, V. V., Biochimica Biophysica Acta, 839 (1985) 57-65] and of α1-thiol proteinases [Otsuka, S, and Yamanaka, T. (Eds), “Metalloproteins-Chemical Properties and Biological Effects” in “Bioactive Molecules”, Kodansha Ltd, Tokyo (1988), pp 18-45] from human plasma using immobilized Zn2+-IDA. An extension of the use of IDA-based IMAC procedures, viz. the purification of recombinant proteins using immobilized Ni2+-nitrilotriacetic acid (Ni2+-NTA) [Hochuli, E., Bannwarth, W., Döbeli, H. and Stuber, D., Bio/Technology, 6 (1988) 1321-1324] (NTA being a structural homologue of IDA), relies on the incorporation at the gene level of a polynucleotide sequence corresponding to a poly-histidine peptide, typically hexa-His, which confers on the protein a higher affinity for binding to immobilized Ni2+-NTA chelating complex, thus enabling the protein to be selectively retained on this IMAC sorbent. In the pre-sent application, the terms “sorbent” and “adsorbent” are used primarily to denote a functionalized polymer substrate (polymer substrate with ligand immobilized thereto) with coordinatively bound metal ion(s), although these terms are also occasionally employed to denote a functionalised polymer substrate without metal ion(s) bound thereto.
As will be noted from the above description of applications of IDA- and NTA-based IMAC systems, an alternative means of altering protein binding selectivity with IMAC systems is through variation in the structure of the chelating ligate. In recent years however, only a handful of new IMAC chelating ligates have been introduced. These include systems based on the bidentate chelators aminohydroxamic acid (AHM) and 8-hydroxyquinoline (8-HQ) [Zachariou, M., Traverso, I., Spiccia, L. and Hearn, M. T. W., Journal of Physical Chemistry, 100 (1996) 12680-12690]; carboxymethylaspartic acid (CM-ASP) which has a higher affinity for Ca2+ than IDA [Porath, J., Trends in Analytical Chemistry, 7 (1988), 254-256; Mantovaara, T., Pertofz, H. and Porath, J., Biotechnology Applied Biochemistry, 11 (1989), 564-569]; ortho-phosphoserine (OPS), which is able to chelate “hard” metal ions such as Fe3+, Al3+, Ca2+ and Yb3+ due to the participation of the phosphate group [Zachariou, M., Traverso, I. and Hearn, M. T. W., Journal of Chromatography, 646 (1993), 107-115]; and other tridentate ligates, such as (2-pyridylmethyl)aminoacetate (CPMA), dipicolylamine (DPA) and cis- or trans-carboxymethyl-proline [Chaouk, H., Middleton S., Jackson W. R. and Hearn, M. T. W., International Journal of BioChromatography, 2 (1997) 153-190; Chaouk, H. and Hearn, M. T. W., Journal of Biochemical and Biophysical Research Methods, 39 (1999) 161-177], tetradentate ligands, such as nitrilotriacetic acid (NTA) [Hochuli, E., Bannwarth, W., Dobeli, H., Gentz, R. and Stuber, D. Bio/Technology, 6 (1988) 1321-1325], which have higher affinities for M2+ ions than IDA due to their quadridentate nature, exhibit lower protein binding association constants due to the loss of one coordination site compared to the IDA-type tridentate ligates; and pentadentate ligands, such as tetraethylenepentamine (TEPA) [Hidaka Y., Park, H. and Inouye, M., FEBS Letters, 400 (1997) 238-242] or N,N,N′-tris(carboxymethyl)ethylene-diamine (TED) [Porath, J., Protein Expression & Purification, 3 (1992) 263-281], which coordinate metal ions via five donor atoms (i.e. two nitrogen atoms of primary amine groups and three nitrogen atoms of secondary amine groups in the case of TEPA, and two nitrogen atoms of secondary amine groups and three oxygen atoms from the three carboxylic groups in the case of TED).
Significant leakage of metal ions has been observed with immobilized metal ion iminodiacetic acid chelate (im-Mn+-IDA) systems when using relatively mild elution conditions in the chromatographic process [Oswald, T., Hornbostel, G., Rinas, U. and Anspach, F. B., Biotechnology Applied Biochemistry, 25 (1997) 109-115; Kagedal, L. in Protein Purification (eds. J. C. Janson and L Ryden) VCH Publishers, New York (1989), pp 227-251]. Thus, in addition to the issue of selectivity modulation, an additional motivation for the development of new classes of chelating ligates has been a need for achieving significant increases in the metal ion stability constants compared to the IDA-based or NTA-based systems which have hitherto been employed [Zachariou, M., Traverso, I., Spiccia, L. and Hearn, M. T. W., Analytical Chemistry, 69 (1996) 813-822].